Chronic hepatitis C is a major unsolved public health problem that afflicts 4 million people and results in 10,000 deaths per year in the USA. Although more than 50 percent of patients eventually succumb to liver failure and/or hepatocellular carcinoma, this frequently takes 20-30 years to develop. This provides a large window for the treatment and prevention of the long-term sequelae of this chronic infection. Unfortunately, current treatments for chronic HCV remain relatively ineffective and frequently have side effects. The hepatitis C virus NS5B polymerase (or RDRP) has been an understudied target for the development of new hepatitis C therapeutics. The primary goal of this work is to further characterize a novel cell-based HCV NS5B polymerase system and use it to identify small molecule inhibitors of NS5B polymerase that may lead to new hepatitis C therapeutics. Hypothesis: This novel mammalian cell-based system will provide the means for identifying and studying small molecules that selectively inhibit the HCV NS5B polymerase and can provide new treatment options for chronic hepatitis C. Specific Objectives: To identify small molecules that inhibit NS5B polymerase, particularly those requiring metabolic activation, using a mammalian cell-based system. To further develop the mammalian cell-based assay so that it has the ability to identify an even broader category of NS5B polymerase inhibitors within mammalian cells. Determine the specificity and toxicity of inhibitors of NS5B polymerase using in vitro assays of mammalian DNA and RNA polymerases, and mammalian cell toxicity assays. PROPOSED COMMERCIAL APPLICATION The market for an effective therapy for chronic hepatitis C is enormous. Approximately 4 million people in the USA have chronic hepatitis C and the world-wide incidence of chronic HCV is estimated at 1.5-2.5 percent. A patent on the mammalian cell NS5B-template/reporter system has been filed by Emory University School of Medicine and licensed to Triangle Pharmaceuticals.